Nucleus export reporter system

ABSTRACT

The present invention relates to reporter systems for RNA export, methods for searching for molecules which influence RNA export, and a method, based on these methods, for detecting a viral infection.

[0001] The present invention relates to reporter systems which permitthe identification of proteins, signal sequences and/or active substancecandidates which regulate the export of RNAs (ribonucleic acids) and, inparticular, viral RNAs from the cell nucleus of eukaryotic cells.

[0002] A wide variety of viruses depend on active export of theirincompletely spliced transcripts from the cell nucleus of the infectedhost cell. This can either take place by use of an RNA signal in ciswithin the viral transcripts (constitutive transport elements), or takesplace with the aid of viral proteins. Cis-active transport elements areused for example from MPMV-CTE (Mason-Pfizer monkey virus constitutivetransport element), SRV-CTE (simian retrovirus constitutive transportelement), hepatitis B virus PRE (posttranscriptional regulatory element)and HSV (herpes simplex virus) (within the TK (thymidine kinase) gene).These RNA elements recruit cellular factors and export pathways in orderto enable nuclear export of the viral transcripts. An alternativepossibility is for nuclear export also to be mediated by an exportfactor which specifically binds to a target sequence within the viraltranscripts and transports the latter into the cytoplasm throughinteraction with cellular factors. Thus, for example, Ad-5 transcriptsare exported with the aid of the 34K and E4orf6 proteins, EBV(Epstein-Barr virus) transcripts with the aid of the EB2 protein,herpes-virus saimiri transcripts with the aid of the ORF 57 geneproduct, HSV (herpes simplex virus) transcripts with the aid of the ICP27 protein, HTLV-I and II (human T-cell leukemia virus I and II)transcripts with the aid of the Rex proteins, EIAV (equine infectiousanemia virus), SIV (simian immunodeficiency virus) and HIV-1 and HIV-2(human immunodeficiency virus 1 and 2) transcripts with the aid of theRev proteins.

[0003] Nuclear export which has been investigated best is that of lateHIV-1 transcripts mediated by HIV-1 Rev. Like all lentiviruses HIV-1depends on a plurality of genes being activated from only one proviraltemplate and being expressed in a fixed time sequence. Different genesare generated from a primary transcript which is only ˜9 kB in size byalternative splicing events and other regulatory mechanisms taking placeat the RNA level. These viral transcripts can be divided into 3 classeson the basis of their size: ˜9 kB unspliced (gag, pol), ˜4 kB singlyspliced (env, vif, vpr, vpu) and ˜2 kB multiply spliced (rev, tat, nef)RNAs.

[0004] Besides the occurrence of incompletely to multiply splicedtranscripts, it is additionally possible to observe a time sequence inthe expression of these different RNA species. Thus, only the multiplyspliced ˜2 kB RNAs, and their gene products Rev, Tat and Nef, aredetectable in the early phase of replication in the cytoplasm of theinfected cells. Only after a time lag do the unspliced (˜9 kB) andsingly (˜4 kB) spliced transcripts and their gene products Gag, Pol andEnv then also appear therein. However, the singly and unsplicedtranscripts can never be detected in the cytoplasm of cells infectedwith viral mutants lacking an active Rev protein. The unspliced andsingly spliced transcripts then accumulate in the nucleus (see FIG.A-4), and the late structural proteins (Gag, Env) and enzymes (Pol)translated from them cannot be formed. The viral Rev protein is thusessentially involved in the time-regulated expression of the viralgenes.

[0005] HIV-1 Rev, just like the RNA transport molecules mentioned above,are shuttle proteins which transport viral RNAs via the interaction withan RNA target sequence located within viral transcripts the latter outof the nucleus into the cytoplasm. Thus, HIV-1 Rev binds specifically inthe nucleus to its RNA target structure RRE, the Rev-responsive element(see FIG. 5 A/B). This region, which is 351 nucleotides (Nt) long, islocated within the Env reading frame and is thus a constituent of allunspliced and singly spliced transcripts. This ribonucleoprotein (RNP)complex is subsequently exported out of the cell nucleus via interactionwith cellular factors. A leucine-rich sequence located at the C terminusis necessary for this and, as nuclear export sequence (NES), mediatesnuclear translocation of the Rev protein through use of cellularmechanisms (Pollard and Malim, 1998).

[0006] The reason why the late transcripts remain in the nucleus in theabsence of Rev, which is a necessary condition for the Rev-dependenceand thus the time-regulated expression of Gag, Pol and Env, is stillcontroversial. In principle there are two main alternative ideas aboutthe nuclear retention of late transcripts.

[0007] It is assumed that a cellular transcript can leave the cellnucleus only when the splicing process is entirely complete, or allactive splice sites have been deleted from the primary transcript. Thelate viral transcripts are intron-containing, only incompletely splicedpre-mRNAs which are transported into the cytoplasm with the aid of Revand RRE. The influence of the cellular splicing machinery on the nuclearretention of the late transcripts was therefore investigated at an earlydate (Mikaelian et al., 1996; Kjems et al., 1991; Kjems and Sharp, 1993;Chang and Sharp, 1989; Powell et al., 1997; Lu et al., 1990; O'Reilly etal., 1995). The presence of splice sites differing in activity appearsto make the splicing process only suboptimal in the case of HIV-1transcripts. Several groups have therefore hypothesized that Rev makesit possible for transcripts which are retained within the splicingmachinery through the formation of inefficient splicing complexes to beexported.

[0008] However, contrary to this it has been possible to show thatexpression of the late HIV-1 genes such as, for example, Env remainsrepressed even in the absence of active splice sites, and thus theinfluence of the splicing machinery appears to be more indirect(Nasioulas et al., 1994). This is why so-called inhibitory sequences(INS) or cis-active repressor elements (CRS) within the reading frames,which adversely influence expression, have been postulated (Nasioulas etal., 1994; Olsen et al., 1992; Schwartz et al., 1992b; Maldarelli etal., 1991). However, these repressor sequences which are located insidethe coding mRNA have no common sequence motif like, for example, theAUUUA instability motif inside the 3′-UTR of the unstable GM-CSF mRNA(Chen and Shyu, 1995), but are conspicuous only by their high A/Ucontent throughout. Thus, fusion of the postulated INS-containingfragments from reading frames of late genes (such as Gag and Env) to aCAT reporter system resulted in a reduced reporter activity (Cochrane etal., 1991; Rosen et al., 1988; Schwartz et al., 1992b). It was possibleto abolish again in part this reduction in the expression of Gag and Polin part by multiple silent point mutations within the wobble positions(Schwartz et al., 1992a; Schneider et al., 1997). The unspliced andsingly spliced HIV-1 mRNAs thus appear to have cis-active repressorelements which are either deleted by multiple splicing or overcome by aRev/RRE-mediated nuclear export.

[0009] There is great medical interest in molecules which modulate, inparticular inhibit, the export of RNA, in particular viral RNA, from thecell nucleus. For example, HIV-1 Rev is an essential factor duringreplication of HI viruses both in cell culture and in vivo. (Feinberg,Jarrett, 1986; Iversen, Shpaer, 1995; Sodroski, Goh, 1986). This makesRev an attractive target for therapeutic agents with antiviral activity.

[0010] Rev-sensitive reporter systems can be used for testing activesubstances which suppress a Rev function and thus prevent productivereplication of HIV-1. A wide variety of systems has been used to datefor evaluating the Rev-dependent HIV-1 gene expression. These extendfrom mutated provirus constructs (Borg et al., 1997; Malim and Cullen,1993) via chimeric Rev-sensitive β-globin genes (Chang and Sharp, 1989;Mikaelian et al., 1996) to subgenetic fragments fused to reporter genes(Schwartz et al., 1992a; Schwartz et al., 1992b).

[0011] However, the systems used to date are not suitable for thehigh-throughput testing (HTT) of therapeutic agents with aRev-inhibiting effect, or for the isolation of specific inhibitors ofthe Rev function from a randomized gene library: working with viralsystems or provirus constructs capable of replication requires S3 safetylaboratories, and elaborate and costly (p24 capture ELISA) detectionmethods which do not permit HTT per se or make it unattractive. ComplexRev-dependent β-globin reporter systems are suitable only for basicresearch, and require special, demanding laboratory methods (Northernblot, RNA protection assay), which likewise preclude HTT. Reportersystems based on a specific enzymatic reaction for detectingRev-sensitive gene expression permit HTT at least in some cases. Thus,it has been possible to convert expression of the chloramphenicolacetyltransferase (CAT) gene into Rev dependence by placing the CATreading frame together with the RRE region inside an intron sequence(Luo et al., 1994; Iacampo, Cochrane, 1996; Hope, Huang, 1990), Thisreporter construct (pDM128 and derivatives) has a number ofdisadvantages, however:

[0012] (a) The assay cannot be used on a single cell basis. Thus cellscannot be separated by FACS but must necessarily be lysed for analysis.

[0013] (b) Simple, easily automatable, optical testing of a Rev actionis not possible.

[0014] (c) Unspliced CAT RNAs can be detected in the cytoplasm of cellstransfected with pDM128 even in the absence of Rev, i.e. a Revdependence is incorrectly simulated in the assay even in the absence ofRev.

[0015] (d) The presence of an intact cellular splicing machinery is aprecondition for the assay. Molecules which influence splicing processesare incorrectly classified as modulators of RNA export in the assay.

[0016] It was therefore an object of the present invention to provide amethod for detecting RNA export from the cell nucleus of a eukaryoticcell which does not have the abovementioned disadvantages of the priorart.

[0017] This object is achieved by a method comprising the steps:

[0018] (a) provision of a nucleic acid which codes for a reporterprotein and whose transcript is exported from the cell nucleus dependingon the presence

[0019] (i) of a cis-active RNA export signal in operative linkage withthe nucleic acid and

[0020] (ii) of a trans-active factor and, where appropriate,

[0021] (iii) of a functional 5′ splice donor in the absence of afunctional 3′ splice acceptor,

[0022] (b) introduction of the nucleic acid into the cell nucleus of thetarget cell so that it is present therein in operative linkage with atranscription control sequence, and that on transcription of the nucleicacid there is generation of a transcript in which the segment of thetranscript coding for the reporter protein cannot be subjected to anysplicing process,

[0023] (c) transcription of the nucleic acid and

[0024] (d) determination of whether the resulting transcript is exportedfrom the cell nucleus.

[0025] The presence of the 5′ splice donor must not lead to a splicingevent being possible as in the case of the constructs of Lu et al.Accordingly, the 5′ splice donor must not be confronted by a 3′ spliceacceptor or at least any efficiently utilized splice acceptor.

[0026] The cis-active RNA export signal may be a previously known RNAexport signal. In this case, the RNA export signal is preferablypositioned in relation to the reporter gene in analogy to the naturaloccurrence of the export signal. Thus, for example, in the case of theRev-responsive element of HIV-1 the signal is cloned downstream of thereporter gene. Conversely, the export signals of adenoviruses arepreferably disposed upstream of the reporter gene.

[0027] The method of the invention is suitable for the identification ofunknown RNA export signals. For this purpose, the reporter gene isoperatively linked in the nucleic acid to a library of gene fragments,and constructs which make RNA export possible are sought. The genefragment library preferably comprises genes or gene fragments from donororganisms or viruses known to have efficient RNA export signals. If itis intended specifically to search for RNA export signals which requirethe presence of viral trans-acting factors, these must likewise beprovided in the cell.

[0028] It is conversely possible, if a cis-active export signal is knownand a trans-active factor is to be identified, to use a construct asdescribed above to search for such a factor. In this case, the nucleicacid construct comprises both the reporter gene and the cis-activesignal. It is then possible to use the reporter system described aboveto search in expression libraries for trans-active factors. Thetrans-active factors are preferably viral adaptor molecules whichmediate an interaction with the cellular RNA export machinery, but theymay also be cellular molecules. Such a trans-active factor includes forthe purposes of this application both a single protein and a proteincomplex.

[0029] If all the cis- and trans-acting factors necessary for RNA exportare provided, the system is suitable for identifying molecules whichinfluence RNA export. The molecule which influences RNA export may be,for example,

[0030] (a) a small molecule, typically having a molecular mass of lessthan 2 000 Da,

[0031] (b) DNA or RNA, derivatives or mimetics thereof, which act at thenucleic acid level or interact with proteins involved in RNA export, or

[0032] (c) a peptide, a modified peptide, protein or modified proteinwhich interacts with nucleic acids or interacts with proteins involvedin RNA export.

[0033] An example of a small molecule as in (a) is leptomycin B, a knowninhibitor of Rev activity. An example of a nucleic acid as in (b) arethe known RRE decoys and an RNA intramer. An example of a protein as in(c) is the transdominant-negative Rev mutant RevM10. It is possible inparticular for the nucleic acids to be derived from a gene library orfor the proteins to be gene products of the genes of a gene library. Inthese cases, the advantage of the system of the invention, of dispensingwith lysis or fixation of the cells, is very particularly evident.

[0034] The activity of leptomycin B and of the transdominant-negativeRev mutant RevM10 were detectable with the reporter system of theinvention (with hivGFP as reporter protein). The produced Rev-sensitivereporter system is thus suitable for detecting inhibition of viral geneexpression and thus as reporter system for identifying therapeuticagents with antiviral activity and acting on viral nuclear export.

[0035] A further substantial improvement of the reporter system of theinvention compared with the previously disclosed CAT system is theavoidance of splicing processes. In the CAT system, RNA is exported intothe cytoplasm in every case. Only in the presence of a trans-activefactor such as, for example, Rev does the latter also contain an intronon which the reporter gene is encoded. By contrast, in the reportersystem of the invention there is no need for a splicing event to takeplace. In the absence of cis- or/and trans-active signals, the RNA isjust not exported into the cytoplasm in amounts which allow theaccumulation of detectable amounts of RNA.

[0036] This can be achieved, for example, by RNA-destabilizing sequencemotifs such as, for example, AUUUA which occurs in the RNA for GM-CSF,or preferably by a choice of codons which reduces the metabolicstability of the RNA. Constructs suitable for reporter systems of theinvention can be generated for example by choosing the codondistribution like that occurring in viral exported RNA. A choice ofcodons to be used preferably in this connection is one like that usedleast frequently or second-least frequently in mammalian cells (Ausubelet al., 1994), even more preferably the choice of codons is adapted tothat of late HIV-1 genes, and even more preferably table 1 is used forproducing the Rev-dependent reading frame.

[0037] For example, the choice of codons of the constitutivelyexpressing gene for green fluorescent protein (GFP) was adapted to thechoice of codons like that to be found in late HIV-1 genes. For thispurpose, the amino acid sequence of the GFP gene product wasback-translated into a synthetic GFP-encoding reading frame using theHIV-1 Gag choice of codons. This reading frame, called hivGFP, was thenconstructed as fully synthetic reading frame using long oligonucleotidesand a stepwise PCR. In addition, the authentic 5′-UTR of the Gag readingframe was put in front of the hivGFP reading frame, and the RRE wasattached 3′ and cloned into an expression vector. The produced hivGFPvector proved to be completely dependent on the presence of the Revprotein in the expression of the autofluorescent GFP. In the absence ofthe 5′-UTR, RRE or Rev it was not possible to detect any expression ofthe green fluorescent reporter. The initial GFP gene, which was adaptedin its choice of codons to mammalian genes (huGFP), by contrast provedto be independent of Rev, RRE or the 5′-UTR in its expression.

[0038] However, instead of adapting the choice of codons as accuratelyas possible to the choice of codons of late HIV genes, it is alsopossible merely to increase the AT content. An AT content of >50% ispreferably aimed at. Increasing the AT content or adapting the codonusage preferably takes place by silent mutations or by mutations whichdo not destroy the activity of the reporter protein. The choice ofcodons need not be adapted if the A/T content of said gene is alreadymore than 50%. Genes with a codon usage differing from the wild type canbe produced as indicated in the example for example from longoligonucleotides and with a stepwise PCR.

[0039] The reporter protein preferably used is a fluorescent protein,because of the particularly simple readability and the suitability forhigh-throughput tests. Examples of autofluorescent reporter proteins arethe green fluorescent protein GFP, the blue fluorescent protein BFP, thered fluorescent protein RFP, the yellow fluorescent protein YFP, orderivatives of these autofluorescent proteins which display increasedfluorescence, such as the enhanced green fluorescent protein eGFP, theenhanced blue fluorescent protein eBFP, the enhanced red fluorescentprotein eRFP or the enhanced yellow fluorescent protein eYFP (Clontech).

[0040] Instead of using fluorescent proteins it is also possible to useother proteins as long as their activity is easily detectable. Examplesare the gene for luciferase LUC, the gene for alkaline phosphatase AP,the gene for secretory alkaline phosphatase SEAP or the gene forchoramphenicol acetyltransferase CAT.

[0041] Immunologically detectable proteins are likewise suitable asreporter proteins. It is sufficient for rapid immunological detection ofthe gene product, of parts of the gene product or of epitopes to bepossible. A frequently used example is the influenzae Flag-tag.

[0042] Proteins capable of positive or negative selection are alsosuitable as reporter proteins. For example, the neomycin-resistance geneprevents a translation block caused by G418 and thus death of the cell.In HAT (hypoxanthine, aminopterin, thymidine) medium, in which de novopurine and pyrimidine biosynthesis is blocked, the activity of thymidinekinase is essential. Conversely, it is possible to select for cellsdeficient in thymidine kinase by propagating the cells inbromodeoxyuridine. Likewise, the enzymic activity of the herpes viralthymidine kinase (TK) brings about the death of TK-expressing cells inthe presence of acyclovir. Further examples of markers capable ofpositive and negative selection are adenine phosphoribosyl transferase(APRT), hypoxanthine-guanine phosphoribosyl transferase (HGPRT), anddihydrofolate reductase (DHFR). Azaserine is used for positive, and8-azaguanine for negative, selection for APRT and HGPRT. In the case ofDHFR, methotrexate is used for selection for the marker, and [³H]dUrd isused for selection against the marker. All the marker systems mentionedare described in detail by Kaufmann (Kaufmann, 1979).

[0043] Finally, the reporter gene may be a regulatory gene which, afterits expression in a cell as molecular switching molecule, switches theexpression of other genes on or off. An example of such a regulatorygene which can be used is a transcription factor.

[0044] In all methods in which it is not intended to search for acis-active RNA export signal, the nucleic acid construct alreadycontains a cis-active RNA export element.

[0045] This may be may be a so-called constitutive transport element. Noviral proteins are necessary for the export for the nuclear export ofRNAs which harbor such a constitutive transport element; the virusmerely utilizes cellular mechanisms which are already present. Examplesof such cis-active RNA export elements are MPMV CRE, RSV CTE or SRV CTE.

[0046] Many viral cis-active RNA export elements are, however, dependenton viral adaptor proteins which mediate the interaction with thecellular export machinery. In the case of HIV, the RRE (Rev responsiveelement) is used as cis-active RNA export element. This signal isrecognized by the viral protein Rev which contains a leucine-richsequence of hydrophobic amino acids which acts as nuclear export signal(NES). The Rev receptor in nuclear export is Crm1, which is also calledexportin 1. Interaction of the Rev receptor with Crm1 can be impaired byleptomycin B. It is thought that the Rex/RxRRE system of HTLV-I andHTLV-II functions analogously to the Rev/RRE system of HIV-1, HIV-2 andSIV.

[0047] The RNA export signal for the RNA export systems describedhereinbefore is preferably located downstream of the structural genes.However, it is also possible to use RNA export signals from viruses inwhich the RNA export signals are located upstream of the structuralgenes, as occurs for example in adenoviruses. In this case, the nuclearexport sequence is preferably located upstream of the reporter gene inthe RNA constructs too.

[0048] The invention further relates to a DNA sequence which codes for areporter protein and is operatively linked to a cis-active RNA exportelement and, where appropriate, to a functional 5′ splice donor in theabsence of a functional 3′ splice acceptor, where

[0049] (a) the DNA sequence coding for the reporter protein is modifiedat the nucleic acid level compared with the wild-type sequence,

[0050] (b) RNA export for the modified DNA sequence takes placedependent on the cis-active RNA export element, and

[0051] (c) essentially no RNA export for the wild-type DNA sequencetakes place dependent on the cis-active RNA export element.

[0052] The wild type means in this connection either the wild type inthe usual sense or else a gene which is optimized for expression in thecorresponding host cell, such as, for example, “humanized GFP”. Thedependence of the modified reporter RNA on the cis-active RNA exportelement is achieved by the methods described above.

[0053] Transcription of the corresponding RNA sequence is controlled bya transcription control sequence. The transcription control sequence maycomprise a constitutive or inducible promoter. Examples of constitutivepromoters which can be used are viral promoters from CMV, SV 40 or fromadenovirus or cellular promoters such as the actin promoter or celltype-specific promoters such as the MHCII promoter. Suitable induciblepromoters are tetracycline-dependent promoters (Tet on/off system), heatshock promoters, metallothionein promoters or promoters which can beinduced by glucocorticoids, such as the promoter in mouse mammary tumorvirus (MMTV) LTR (Kaufmann, 1979). An appropriate polyadenylation signalis attached to the 3′ end of the DNA for the polyadenylation of the RNAtranscripts. The statements made above apply to the choice of thereporter protein and of the cis-active RNA export signal. It isappropriate in some circumstances to provide a trans-active factor andthe reporter construct on the same plasmid.

[0054] The present invention further relates to eukaryotic cells, morepreferably mammalian cells, most preferably human cells, which aretransformed with a DNA construct as described above, where the DNAconstruct is present in a form capable of transcription. The DNAconstruct may for example exist episomally or be stably integrated intothe chromosome. It is moreover possible for one or more copies to bepresent in the cell. If an RNA export sequence which is active only inthe presence of a viral export factor is used, it is possible bytransfection or infection of the cell with expression libraries tosearch for viral factors which interact with the RNA export sequence oract further downstream in the RNA export pathway, if such factors arenot yet known. If the RNA export activity is successfully reconstituted,it must be possible to measure an increase in the expression of theRev-dependent gene by at least 2.5-fold, preferably 5-fold, morepreferably 8-fold or more.

[0055] If the factors are already known, they are preferably madeavailable in the cell, usually in trans. The invention therefore relatesto the production of stable cell lines which, besides the exportconstruct described above, also harbor the gene for an additional viralexport factor integrated chromosomally or episomally in them. It ispossible to use according to the invention any eukaryotic cell in whichthe factors necessary for RNA export are present in concentrations whichcorrespond approximately to the natural infection model. Except for theinfection model described hereinafter, it is unnecessary for the cellsto be permissive for replicative infection with the viral system to beinvestigated. Examples of eukaryotic cells which can be used are H1299cells, HeLa cells, HEK cells (human embryonic kidney cells).

[0056] The viral export factor is preferably Rev from HIV-1, HIV-2, SIVand EIAV or Rex from HTLV-I and HTLV-II, or the 34K and E4orf6 proteinsfrom adenoviruses, or EB2 proteins from EBV, or ORF 57 gene productsfrom herpes virus saimiri, or ICP 27 proteins from HSV.

[0057] The present invention further relates to the production of stablecell lines which, besides the Rev-dependent gene, additionally compriseone or more constitutively expressing genes. It is possible bydetermining the rate of expression of these genes or the amount of therelevant protein products to distinguish whether an active substances tobe tested specifically affects the expression dependent on the exportfactor provided in trans, or only generally blocks cellular expression.The reporter gene whose expression is regulated by the viral exportfactor, and the constitutively expressed protein must be detectable bydifferent methods. If both proteins are fluorescent, they must thereforediffer in the wavelength of the exciting light or in the wavelength ofthe emitted light. However, it is also possible to use other enzymaticmethods or the recognition of another immunological epitope.

[0058] The DNA reporter construct and a cell which can be transfectedwith the DNA or a cell which is already transfected with the DNAconstruct can be provided as reagent kit for investigating RNA exportprocesses. The reagent kit preferably also comprises leptomycin B,because it is possible with this substance specifically to block the RNAexport pathway utilized for example by HIV Rev-dependent RNA constructs(Otero, 1998).

[0059] The present invention further relates to a method for detecting aviral infection which has taken place, more preferably a retroviralviral infection, most preferably a lentiviral viral infection. Thedetection is based on the fact that a viral export factor necessary fornuclear export is present in infected cells but not in uninfected cells.If HIV infection is involved, the viral RNA export factor is HIV Rev,and in the case of HTLV infection the viral RNA export factor is HTLVRex.

[0060] Because patients' plasma is easily available, it is appropriateto detect the infection through detecting infectious viruses in theplasma. In this embodiment, a cell which is transfected with thereporter construct, preferably carries the reporter construct stably initself, particularly preferably has the reporter construct stablyintegrated into the chromosome, is brought into contact with thepatient's plasma. The cell is infected if viruses are present in theplasma. The necessary, trans-active RNA export factor is provided inthis way, and RNA export can be detected.

[0061] Detection of infection is also possible even if viruses are nolonger detectable in the plasma but, nevertheless, cells are infectedwith the virus. In this case the infection must originate from thesecells. An alternative possibility is for the cells also be transfectedwith the reporter construct.

[0062] The infection can, if the reporter gene is chosen suitably, bedetected by fluorescent bioanalysis, where appropriate through theautofluorescence of the reporter gene product, preferably byspectroscopy, preferably by fluorescence microscopy, more preferably byFACS analyses. This detection can take place manually or completelyautomatically in a high-throughput testing.

[0063] An infection can be detected through detection of the reportergene dependent on the viral export factor, in particular Rev,additionally via the enzymatic activity of the chosen reporter geneproduct, such as the phosphatase activity of SEAP, the acetyltransferaseactivity of the CAT gene product, the luciferase activity of theluciferase gene. This detection can take place manually or completelyautomatically in a high-throughput testing.

[0064] However, the antigenic properties of a reporter protein or of oneor more epitopes of the chosen reporter protein can also be utilized.Detection then takes place by suitable immunological methods such asELISA, immunofluorescence, immunoblot, FACS of immunologically labeledcells. It is possible manually or completely automatically in ahigh-throughput testing.

[0065] It was possible to show in particular that cells transfected withthe Rev-sensitive hivGFP together with an infectious proviral HIV-1clone (HX10) likewise exhibit a green fluorescent reporter geneactivity. The produced Rev-sensitive reporter system is thus suitablefor detecting HIV-1 gene expression and thus as detection of infection.

[0066] A further detection of the RNA export detection of Lu et al. isthe necessity to have to lyse or fix the cells to detect the reporterprotein or the activity of the reporter protein. The invention thereforefurther relates to a method for detecting RNA export from the cellnucleus, in which a reporter protein that can be detected without lysisor fixation of the cells is used. A fluorescent protein for example issuitable for this purpose. However, it is likewise also possible to usea suitable selection marker. The advantage of this method compared withthe prior art is the possibility

[0067] (a) of being able to transfect or infect the cells with a genelibrary

[0068] (b) of being able to purify the genes which bring aboutmodulation of the RNA export from the cell nucleus by cultivating thecells and isolating the nucleic acid.

EXAMPLES 1. Production of a Rev-Dependent GFP Gene

[0069] The intention was to construct artificially the reading frame ofthe green fluorescent protein (Gfp) gene using a choice of codons likethat to be found in HIV-1 structural genes. For this purpose, the aminoacid sequence of the Gfp gene was translated into a correspondingnucleotide sequence. This was carried out with the aid of the gcgcommand “backtranslate” using an appropriate matrix as described intable 1. Further cleavage sites were inserted for subcloning and forattaching further sequence elements within untranslated regions. Anexact sequence, including the cleavage sites used, is indicated in SEQID No. 1. The sequence produced in this way was produced as completelysynthetic gene using synthetic oligonucleotides and a previouslydescribed method {Zolotukhin, Potter, 1996}. FIG. 1 depicts a comparisonof the hivGFP (choice of codons derived from HIV structural genes) andhuGFP (choice of codons derived from mammalian genes). The GFP-encodingDNA fragment (“hivGFP”) produced in this way was placed under thetranscriptional control of the cytomegalovirus (CMV) earlypromoter/enhancer (”pc-hivGFP”) in the expression vector pcDNA3.1(+)(Stratagen, Heidelberg) using the KpnI and XhoI cleavage sites. Toproduce an analogous GFP expression plasmid whose choice of codons was,however, adapted to the human system, the coding region of the humanizedGFP gene (huGFP) was amplified from a commercially obtainable vector bymeans of a polymerase chain reaction (PCR) using the oligonucleotideshu-1 and hu-2 and cloned into the expression vector pcDNA3.1(+)(Stratagen, Heidelberg) likewise using the KpnI and XhoI cleavage sites(“pc-huGFP”).

[0070] As explained in the description, an isolated (efficientlyutilizable) splice donor (SD) must be put in front of the coding regionin order to achieve Rev dependence of the hivGFP reporter. The HIV-1untranslated region (UTR) within the late HIV-1 transcripts containssuch an SD. This region was amplified by means of PCR using theoligonucleotides utr-1 and utr-2 from proviral HIV-1 DNA (HX10, see(Ratner et al., 1987)) and cloned directly 5′ in front of the ATG of theGFP-encoding reading frame of the pc-huGFP and pc-hivGFP constructsusing the KpnI and NcoI cleavage sites. The resulting constructs havebeen referred to hereinafter as “pc-UTR-huGFP” and “pc-UTR-hivGFP”,respectively.

[0071] As explained in the applications, it is necessary to attach tothe coding region an RNA target sequence in order to achieve Revdependence of the hivGFP reporter. This target sequence interacts at theRNA level either with a viral nuclear export protein (in the case ofHIV-1 the Rev protein) or cellular nuclear export proteins. For thisreason, the HIV-1 Rev-responsive element from proviral HX10 DNA wasamplified by means of PCR using the oligonucleotides rre-1 and rre-2 andcloned 3′ behind the GFP-encoding region of the pc-huGFP, pc-UTR-huGFP,pc-hivGFP, pc-UTR-hivGFP constructs using the BamHI and XhoI cleavagesites. The resulting constructs have been referred to hereinafter as“pc-huGFP-RRE”, “pc-UTR-huGFP-RRE”, “pc-hivGFP-RRE”,“pc-UTR-hivGFP-RRE”. In addition, the MPMV constitutive transportelement CTE from proviral MPMV DNA was amplified by means of PCR usingthe oligonucleotides cte-1 and cte-2 and cloned 3′ behind theGFP-encoding region of the pc-hivGFP, pc-UTR-hivGFP constructs using theBamHI and XhoI cleavage sites. The resulting constructs have beenreferred to hereinafter as “pc-hivGFP-CTE”, “pc-UTR-hivGFP-CTE”. All theGFP-encoding constructs produced are depicted diagrammatically in FIG.2.

[0072] For the Norhern blot analyses, additionally the RRE-encodingregion was cloned in antisense orientation to the T7 promoter into thepCR-Script vector (Stratagene, Heidelberg). In addition, the pSP6-actinconstruct was kindly made available to us by F. Schwarzmann's group(IMMH, Regensburg). To provide the viral Rev protein in trans, Prof. J.Hauber (Erlangen) kindly made available a Rev expression plasmid to us.

2. Rev-Dependent GFP Expression of the hivGFP Reporter Requires theChoice of Codons of HIV Structural Genes and the 5′-UTR/SD

[0073] All the cell culture products were from Life Technologies(Karlsruhe). All mammalian cell lines were cultivated at 37° C. and 5%CO₂. The human lung carcinoma cell line H1299 was grown in Dulbecco'smodified Eagle medium (DMEM) with L-glutamine, D-glucose (4.5 mg/ml),sodium pyruvate, 10% inactivated fetal bovine serum, penicillin (100U/ml) and streptomycin (100 μg/ml). The cells were subcultivated in theratio 1:10 after confluence was reached.

[0074] 1.5*10⁶ cells were seeded in Petri dishes (diameter: 100 mm) and,24 h later, transfected by calcium phosphate coprecipitation (Graham andEb, 1973) with 30 μg of indicator plasmid and 15 μg of pc-Rev or 15 μgof pcDNA 3.1 vector. Cells and culture supernatants were harvested 48 hafter transfection. The transfected cells were washed twice withice-cold PBS (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl),scraped off in ice-cold PBS, centrifuged at 300 g for 10 min and lysedin lysis buffer (50 mM Tris-HCl, pH 8.0, 0.5% Triton X-100 (w/v)) on icefor 30 min. Insoluble constituents of the cell lysate were removed bycentrifugation at 10 000 g and 4° C. for 30 min. The total amount ofprotein in the supernatant was determined using the Bio-Rad ProteinAssay (Bio-Rad, Munich) in accordance with the manufacturer'sinstructions.

[0075] The samples were mixed with the same volume of 2× sample buffer(Laemmli, 1970) and heated at 95° C. for 5 min. 50 μg of total proteinfrom cell lysates or half the sample batch from enriched supernatants(B.4.1) were fractionated on a 12.5% SDS/polyacrylamide gel (Laemmli,1970) electrotransferred to a nitrocellulose membrane and analyzed usingthe monoclonal p²⁴-specific antibody 13-5 (Wolf et al., 1990) anddetected by means of a secondary, HRP (horse-radish preoxidase)-coupledantibody and detected by chromogenic staining.

[0076] The reporter constructs were transiently transfected into H1299cells. The expression achieved was analyzed in the presence and absenceof Rev/RRE and of the 5′-UTR/SD. The huGFP expression could not beenhanced either by the Rev/RRE system (FIG. 3A, lanes 3, 4), nor was itsignificantly influenced by the 5′-UTR/SD (FIG. 3A, lane 5). Nor was thecombination of 5′-UTR/SD and Rev/RRE able to convert the syntheticreading frame into Rev dependence (FIG. 3A, lane 6). However, incontrast to this, the hivGFP reporter constructs adapted to the choiceof codons of HIV-1 structural genes behaved completely differently.Thus, no GFP expression was detectable in the absence of the 5′-UTR/SD,irrespective of the presence or absence of the Rev/RRE system (FIG. 3A,lane 7, 8). Only in the presence of the 5′-UTR/SD and of the hivGFPreporter. gene adapted to HIV-1 structural genes was it possible todetect Rev/RRE-dependent expression of the GFP reporter (FIG. 3A, cf.lane 9 with 10).

[0077] Rev-dependent expression based on nuclear export of viral orquasi-viral transcripts should not be attributable by a simplestabilization of the transcripts by the Rev/RRE interaction alone.Although the Rev-M10 mutant can interact with the RNA target sequenceRRE and other Rev proteins (mutated as well as wild type), it cannot beexported from the nucleus due to a defect within the nuclear exportsignal (NES) (Stauber et al, 1995; Kubota et al, 1991). GFP expressioncould not be achieved in the presence of the Rev-M10 protein aftertransfection with the UTR-hivGFP-RRE reporter (FIG. 3B, lane 3).Rev-dependent expression of the hivGFP reporter is thus not attributableto stabilization of the RNA by a Rev/RRE interaction but to thesubsequent nuclear export through wild-type Rev. It was moreoverpossible through the UTR-hivGFP-RRE reporter to achieve GFP expressionin the presence of cotransfected proviral HIV-1 DNA (HX10) (FIG. 3B,lane 2). It is thus possible to detect an HI viral infection by means ofthe Rev formed during viral replication by the hivGFP reporter system.In addition, hivGFP expression using the heterologous CTE nucleartranslocation system likewise proved to be dependent on the presence ofthe 5′-UTR/SD and on the choice of codons used (FIG. 3C). This showsthat the reporter system based on this invention is based on asuperordinate nuclear retention principle which operates irrespective ofthe nuclear export mechanism.

3. Rev-Dependent GFP Expression of the hivGFP Reporter is Based onRev-Mediated Nuclear Translocation

[0078] The experiments summarized in FIG. 3 suggest that there isRev-mediated nuclear export of the UTR-hivGFP-RRE transcripts. In orderto prove this suggestion, the subcellular distribution of theGFP-encoding transcripts was subjected to Northern blot analysis.GFP-encoding transcripts were detected using an RRE-specific probe. Inaddition, the amount and integrity of the β-actin RNA were detected asinternal control of the quality and quantity of the RNA preparation.

[0079] For this purpose, transfected cells were detached bytrypsinization and washed 2× with ice-cold PBS. 1×10⁷ cells werepartially lysed with 175 μl of lysis buffer (50 mM Tris, 140 mM NaCl,1.5 mM MgCl₂, 0.5% NP-40, pH 8.0) on ice for 5 min. The cytoplasmicfraction was separated from the nuclear fraction by centrifugation(300×g, 2 min) and placed on ice. The nuclei were cautiously washed withlysis buffer and again centrifuged (300×g, 2 min). The total DNA wasprepared from the nuclei and from the cytoplasmic fraction in each caseusing the RNeasy kit (Qiagen, Hilden). The RNA preparations were takenup in RNAase-free water and stored at −80° C. until used further.

[0080] The following solutions were used for the Northern blot analysis:SSPE (20x): 175.3 g of NaCl, 27.6 g of NaH₂PO₄ H₂O, 7.4 g of EDTA add 11 1 1 of H₂O (adjust to pH 7.4) MOPS (10x): 83.72 g of MOPS, 8.23 g ofNaAc, 20 ml of EDTA (0.5 N) add 1 1 of H₂O (adjust to pH 7.0) SSC (20x):87.7 g of NaCl, 44.1 g of Na citrate add 500 ml of H₂O (adjust to pH7.0) Denhards 1 g of Ficoll (type 400), 1 g of (100x):polyvinylpyrrolidones, 1 g of BSA (fraction V) add 50 ml of H2OHybridization 12.5 ml of SSPE (20x), 25 ml of buffer: formamide, 2.5 ofDenhards (100x), 2.5 ml of SDS (10%), 20 mg of tRNA (from brewers yeast,Boeringer Mannheim) RNA sample 10.0 ml of formamide, 3.5 ml of buffer:foraldehyde, 2.0 ml of MOPS (5x) RNA loading 50% of gylcerol, 1 mM ofEDTA, 04% buffer: bromophenol blue

[0081] The Northern blot analyses were carried out on the basis of the“Promega” protocol (RNA Applications Guide, Promega Corporation,Madison, USA). 10 μl of RNA preparation were provided with 20 μl ofsample buffer and 5 μl of loading buffer and fractionated on a 1%agarose gel (0.04M MOPS, 0.01M NaAc, 0.001M EDTA, 6.5% formaldehyde, pH7.0). The RNA, gel was blotted by capillary force on a negativelycharged nylon membrane (Boehringer, Mannheim) overnight. The RNA wasfixed on the membrane by UV treatment (1200 kJ for 1 min), and nucleicacids were stained nonspecifically (0.03% methylene blue, 0.3M NaAc).The size standard, and the 18S and 28S RNA were marked and the blot wasdecolorized in water. The membrane was preincubated in 10 ml ofhybridization buffer at 60° C. for 2 h and hybridized overnight afteraddition of a radiolabeled RNA probe. The blot was then evaluated afterstringent washing several times (0.1 SSC, 01% SDS) by exposure on aPhosphor-Imager plate and with the aid of the Molecular Analyst software(Bio-Rad Laboratories, Munich).

[0082] Radioactive antisense RNA probes for specific detection of theRNA were produced by in vitro transcription using the Riboprobe in vitrotranscription system (Promega, Madison, USA), observing themanufacturer's instructions. The radiolabeled nucleotide used was³²P-α-CTP (10 μCi per reaction). The transcription template employed ineach case was 500 ng of linearized plasmid DNA. An RRE-specific RNAprobe was produced by T7-mediated transcription of XhoI-linearizedpc-ERR. A β-actin-specific RNA probe was produced by SP6-mediatedtranscription of EcoRI-linearized pSP6-actin.

[0083] The amounts of cytoplasmic RNA correlated for all the reporterconstructs with the measured GFP expression (FIG. 4, lanes 7-12). In theabsence of the 5′-UTR it was possible to detect only minimal amounts ofhivGFP RNA (hivGFP-RRE) in the nucleus, even when Rev was made availablein trans (FIG. 4, lane 5, 6). In contrast to this, in the presence ofthe authentic 5′-UTR (UTR-hivGFP-RRE), the GFP RNAs adapted to thechoice of codons of HIV structural genes accumulated in the nucleus andwere detectable in large amounts therein (FIG. 4, lane 1).

[0084] Without adaptation of the choice of codons to HIV-1 structuralgenes, the huGFP RNA proved to be stable in the nucleus and wasconstitutively transported into the cytoplasm, even when the 5′-UTR/SDwas placed in front of the coding region. It is thus impossible solelyby placing an efficiently ultilized SD in front to convert anyparticular gene into Rev dependence in the absence of an (efficientlyutilized) SA.

[0085] Rev-dependent GFP expression of hivGFP can be inhibited withtherapeutic agents which block Rev-mediated nuclear export. As describedin the applications, it was possible to use the Rev-dependent hivGFPreporter system described herein for identifying therapeutic agents withantiviral activity, especially those which inhibit the nuclear export ofthe quasi-viral GFP-encoding RNA. Leptomycin B (LMB) and thetransdominant-negative Rev mutant M10 (Rev M10) are the best knowninhibitors of Rev-mediated nuclear export. These established activesubstances should likewise have an inhibitory effect on Rev-dependentexpression of the hivGFP reporter. In order to test this, hivGFPexpression was analyzed in the presence of Rev and LMB or Rev M10.

[0086] For the Rev M10 experiments, 3×10⁵ H1299 cells were seeded in a6-well cell culture plate and, 24 h later, transfected with 5 μg ofreporter plasmid, 2.5 μg of pc-Rev and increasing amounts of pc-RevM10by calcium phosphate coprecipitation. In addition, the transfectionmixture was adjusted in each case to a total of 15 μg of total DNA withpcDNA 3.1 plasmid DNA.

[0087] For the LMB experiments, 3×10⁵ H1299 cells were seeded in a6-well cell culture plate and, 24 h later, transfected with 10 μg ofreporter plasmid and 5 μg of pc-Rev or pcDNA 3.1 plasmid DNA by calciumphosphate coprecipitation. 24 h before harvesting, the medium wassupplemented with 5 nM of LMB. The cells were harvested and GFPexpression was read as described above.

[0088] It was possible even by cotransfection with equimolar amounts ofRevM10 to reduce greatly Rev-dependent expression of the UTR-hivGFP-RREreporter construct (FIG. 5A). It was likewise possible to inhibit onlythe expression of the Rev-dependent GFP reporter through the presence of5 nM LMB, but not the expression of the huGFP reporter (FIG. 5B). It wasthus possible to demonstrate that the hivGFP RNA leaves the cell nucleusby the same nuclear export pathway (CRM1) as late HIV-1 transcripts. Itwas additionally possible to demonstrate that GFP expression of theestablished Rev-dependent GFP reporter can be inhibited by Revinhibitors in the same way as the expression of late HIV-1 genes. Thesimple autofluorescent detection of the GFP reporter thus makes thissuitable for identifying Rev inhibitors with antiviral activity.Rev-dependent GFP expression of the hivGFP reporter can be detected on asingle-cell basis and can be quantified by flow cytometry.

[0089] The autofluorescent properties of GFP permit detection ofRev-dependent expression of the hivGFP reporter system on a single-cellbasis. For microscopic decetion, sterile slides were for this purposeplaced in Petri dishes, 10⁶ H1299 cells seeded thereon and and, 24 hlater, transfected with 30 μg of GFP reporter plasmid and 15 μg ofpc-Rev or 15 μg of pcDNA 3.1(+) by calcium phosphate coprecipitation.After 48 h, the slides were washed 2× with PBS, fixed with 4%paraformaldehyde (10 min) and then stained with DAPI (1 mg/ml) at 37° C.for 1 h. Microscopic detection of the GFP gene product took place withthe aid of an Olympus AX500 fluorescent microscope.

[0090] In the case of the reporter based on huGFP there was detectableGFP activity unaffected by the presence and absence of Rev. By contrast,green fluorescent reporter activity of the GFP was detectable inUTR-hivGFP-RRE-transfected cells only when Rev was cotransfected (FIG.6). Detection of the Rev-dependent GFP reporter is thus possible on asingle-cell basis.

[0091] In order to be able to quantify this better, in addition the GFPactivity of transfected cells was subjected to an FACS analysis. For theanalysis in a flow cytometer, 10⁶ H1299 cells were seeded in Petridishes and, 24 h later, transfected with 30 μg of reporter construct and15 μg of pc-Rev or pcDNA 3.1(+) by calcium phosphate coprecipitation.The transfected cells were detached by trypsinization 48 h later, takenup in ice-cold PBS and subjected to an FACS analysis. The results havebeen represented in a 2-dimensional diagram. For this purpose, thefluorescence intensity (x axis) was plotted against the cell count (yaxis). It was possible to confirm the results of the expression analysesusing the Western blot technique by the FACS analyses. Once again, theRev/RRE system brought about no increase in GFP expression, irrespectiveof the presence or absence of the 5′-UTR (FIG. 7, 2 to 4). Only in theGFP reporter constructs whose choice of codons was adapted to that ofHIV-1 structural genes and simultaneously had a 5′-UTR/SD was itpossible to detect Rev/RRE-dependent GFP reporter activity. In thepresence of Rev and RRE it was possible to increase the GFP activity ofthe hivGFP reporter from a scarcely detectable background activity (2.2%of huGFP) to more than 15 times the level (36.2% of huGFP). The GFPreporter construct produced on the basis of this invention accordinglypermits quantitative detection of Rev-mediated nuclear export on asingle basis and is thus suitable for high-throughput testing.

Sequences

[0092] utr-1: gat cga att ccg acg cag gac tcg gct tgc utr-2: gat ccc atggct ctc tcc ttc tag cct ccg rre-1: gat cgg atc cga gat ctt cag acc tggagg ag rre-2: gat cct cga ggt tca cta atc gaa tgg atc tg hu-1: gat cgaatt caa cca tgg tga gca agg gcg agg ag hu-2: gat cct cga gaa gga tcc tttact tgt aca gct cgt c cte-1: gct agg atc ccc att atc atc gcc tgg aaccte-2: cga act cga gca aac aga ggc caa gac atc

[0093] TABLE 1 Preferred choice of codons for producing a Rev-dependentgene. Amino acid Codon Priority Ala GCA 1 Ala GCT 2 Arg AGG 2 Arg AGA 1Asn AAT 1 Asn AAC 2 Asp GAT 2 Asp GAC 1 Cys TGT 1 Cys TGC 2 End TAG 2End TAA 1 Gln CAG 2 Gln CAA 1 Glu GAG 2 Glu GAA 1 Gly GGG 2 Gly GGA 1His CAT 1 His CAC 2 Ile ATA 1 Ile ATT 2 Leu TTA 1 Leu CTA 2 Lys AAG 2Lys AAA 1 Met ATG 1 Phe TTT 1 Phe TTC 2 Pro CCA 1 Pro CCT 2 Ser AGC 1Ser TCA 2 Thr ACA 1 Thr ACT 2 Trp TGG 1 Tyr TAT 1 Tyr TAC 2 Val GTA 1Val GTG 2

References

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[0095] Borg, K. T., Favaro, J. P., and Arrigo, S. J. (1997). Involvementof human immunodeficiency virus type-1 splice sites in the cytoplasmicaccumulation of viral RNA. Virology 236, 95-103.

[0096] Chang, D. D. and Sharp, P. A. (1989). Regulation by HIV Revdepends upon recognition of splice sites. Cell 59, 789-795.

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FIGURES

[0122]FIG. 1: A sequence comparison of the humanized GFP gene versus thehivGFP gene whose choice of codons has been adapted to that of HIV-1structural genes is depicted. Almost every third wobble position hasbeen replaced mostly by an A or T. This reduces the homology of the tworeading frames to 67.9%. The total AT content thus increases from about37% (huGFP) to about 69% (hivGFP), but without changing the amino acidsequence of the resulting gene products.

[0123]FIG. 2: Diagrammatic representation of all the GFP reporterconstructs produced and used in this study. Open boxes symbolize GFPgenes (hivGFP) adapted to HIV structural genes, and black boxessymbolize GFP genes (huGFP) adapted to mammalian genes. Horizontal linessymbolize untranslated regions. All other symbols are explainedunderneath the image.

[0124]FIG. 3: Expression analysis of the synthetic reading frames. H1299cells were transfected with the stated GFP constructs, and cotransfectedwith the vectors (+) indicated below the images or blank vector (−). GFPproduction was detected by conventional immunoblot analysis. (A) Testingof a Rev-dependent GFP expression. (B) Testing of GFP expression ofUTR-hivGFP-RRE in the presence of Rev, of a mutated form of Rev (RevM10) and of proviral HIV-1 DNA (HX10). (C) Testing of a CTE-mediated GFPexpression of the reporter constructs. Position and molecular weight ofthe GF protein are indicated by an arrow on the right-hand margin.

[0125]FIG. 4: Northern blot analysis and subcellular distribution ofGFP-encoding RNAs. Transfected H1299 cells were partially lysed with0.5% NP-40 buffer, and the nuclei were separated from the cytoplasm bycentrifugation. The total RNA was prepared in each case from the nuclearand cytoplasmic fraction and subjected to a Northern blot analysis. GFPtranscripts adapted to HIV-1 structural genes and adapted to mammaliangenes were detectable simultaneously by means of an RRE-specific probe.The size and position of the GFP RNA is indicated by an arrow. Asinternal control, the RNA of the housekeeping gene β-actin was likewisedetected.

[0126]FIG. 5: Effect of Rev inhibitors on the expression of the GFPreporters. H1299 cells were transfected with the stated GFP constructsand analyzed by Western blotting. (A) To test the effect of thetransdominant Rev mutant (Rev M10) on GFP expression of theUTR-hivGFP-RRE reporter, 5 μg of the reporter were cotransfected with ineach case with 2.5 μg of Rev plasmid and increasing amounts of Rev M10expression plasmid (0; 2.5; 5; 7.5). (B) To test the effect of LMB onGFP expression, the stated constructs were cotransfected in the presence(+) and absence (−) of 5 nM LMB, and Rev(+) and blank vector (−)

[0127]FIG. 6: Immunofluorescence analysis of the huGFP and hivGFPreporter. H1299 cells were transfected with the stated constructs andcotransfected with Rev or blank vector (no Rev) on slides. After 48 h,the cells were fixed and stained with DAPI, and the autofluorescentactivity of the GFP gene product was evaluated in an immunofluorescentmicroscope.

[0128]FIG. 7: Flow cytometry analysis of transfected H1299 cells. 1:Mock, 2: huGFP, 3: UTR-huGFP-RRE, 4: UTR-huGFP-RRE and REV., 5:hivGFP-RRE, 6: hivGFP-RRE and REV, 7: UTR-hivGFP-RRE, 8: UTR-hivGFP-RREand REV. The Y axis indicates the scattered light intensity, and the Xaxis indicates the GFP-related fluorescence. The vertical line dividesthe cell populations into (left) non-fluorescent and measurablefluorescent GFP activity (right). The percentage amounts of cells areindicated at the top in the analyses. The red circles include cellpopulation with high fluorescent GFP activity. The percentage amounts ofthese cells are indicated below in the analyses (marked red).

[0129]FIG. 8: Diagrammatic representation of a Rev-dependent gene. Thesynthetic gene must have a choice of codons which is unusual formammalian genes or a thoroughly high (>50%) A/T content. An untranslatedregion (UTR) which comprises an effective splice donor is positioned 5′from this gene. The viral target sequence must be positioned 3′ fromthis gene. This target sequence may be either a constitutive transportelement such as of the MPMV CTE, or the target sequence of a viral RNAtransport molecule such as of the HIV-1 RRE. Cellular or viral exportfactors then mediate the nuclear export. The Rev-dependent gene is underthe transcriptional control of an inducible (Tet on/off) or constitutive(CMV, SV40) promoter. Polyadenylation of the transcripts is ensured by apolyadenylation signal such as of the BGHpoly(A) signal.

1. A method for detecting RNA export from the cell nucleus of aeukaryotic target cell, comprising the steps: (a) provision of a nucleicacid which codes for a reporter protein and whose transcript is exportedfrom the cell nucleus depending on the presence (i) of a cis-active RNAexport signal in operative linkage with the nucleic acid and (ii) of atrans-active factor and, where appropriate, (iii) of a functional 5′splice donor in the absence of a functional 3′ splice acceptor, (b)introduction of the nucleic acid into the cell nucleus of the targetcell so that it is present therein in operative linkage with atranscription control sequence, and that on transcription of the nucleicacid there is generation of a transcript in which the segment of thetranscript coding for the reporter protein cannot be subjected to anysplicing process, (c) transcription of the nucleic acid and (d)determination of whether the resulting transcript is exported from thecell nucleus.
 2. The method as claimed in claim 1 for identifyingcis-active RNA export elements.
 3. The method as claimed in claim 1 foridentifying trans-active RNA export factors.
 4. The method as claimed inclaim 1 for identifying molecules which influence RNA export.
 5. Themethod as claimed in claim 4, characterized in that the molecule to beidentified is a small molecule having a molecular mass of less than 2000 dalton, is DNA, RNA, derivatives or mimetics thereof, is a peptide,a modified peptide, a protein or a modified protein.
 6. The method asclaimed in any of the preceding claims, characterized in that the RNAexport dependence of the nucleic acid is achieved by a choice of codonswhich reduces the metabolic stability of the reporter RNA.
 7. The methodas claimed in any of the preceding claims, characterized in that the RNAexport dependence of the nucleic acid is achieved by an at least partialadaptation of the choice of codons of the nucleic acid to the choice ofcodons of exported viral RNA.
 8. The method as claimed in any of thepreceding claims, characterized in that the RNA export dependence of thenucleic acid is achieved by an at least partial adaptation of the choiceof codons of the nucleic acid to the choice of codons of HIV-1 (humanimmunodeficiency virus 1).
 9. The method as claimed in any of thepreceding claims, characterized in that the RNA export dependence of thenucleic acid is achieved by increasing the A/T content of the nucleicacid.
 10. The method as claimed in any of the preceding claims,characterized in that the nucleic acid is a synthetically producednucleic acid.
 11. The method as claimed in any of the preceding claims,characterized in that the reporter gene is a gene for a fluorescentprotein.
 12. The method as claimed in claim 11, characterized in thatthe fluorescent protein is GFP (green fluorescent protein), BFP (bluefluorescent protein), RFP (red fluorescent protein), YFP (yellowfluorescent protein), eGFP (enhanced green fluorescent protein), eBFP(enhanced blue fluorescent protein), eRFP (enhanced red fluorescentprotein), eYFP (enhanced yellow fluorescent protein) or hrGFP.
 13. Themethod as claimed in any of the preceding claims, characterized in thatthe gene product of the reporter gene is a protein whose enzymaticactivity is detectable.
 14. The method as claimed in claim 13,characterized in that the gene product is LUC (luciferase), AP (alkalinephosphatase), SEAP (secretory alkaline phosphatase) or CAT(chloramphenicol acetyltransferase)
 15. The method as claimed in any ofthe preceding claims, characterized in that the reporter protein is animmunologically detectable protein.
 16. The method as claimed in any ofthe preceding claims, characterized in that the reporter protein is aselection marker.
 17. The method as claimed in any of the precedingclaims, characterized in that the reporter protein involves a regulatorygene, in particular a transcription factor.
 18. The method as claimed inany of the preceding claims, characterized in that the nucleic acidcomprises a cis-active RNA export element.
 19. The method as claimed inclaim 18, characterized in that the cis-active RNA export element is aconstitutive RNA export element.
 20. The method as claimed in claim 19,characterized in that the cis-active RNA export element is MPMV CTE(Mason Pfizer monkey virus constitutive transport element), RSV CTE(Rous sarcoma virus constitutive transport element) or SRV CTE (simianretrovirus constitutive transport element).
 21. The method as claimed inclaim 18, characterized in that the cis-active RNA export element is anexport element which is recognized by a viral export factor.
 22. Themethod as claimed in claim 21, characterized in that the cis-active RNAexport element is HIV-1 RRE (human immunodeficiency virus 1 Revresponsive element), HIV-2 RRE (human immunodeficiency virus 2 Revresponsive element), SIV RRE (simian immunodeficiency virus Revresponsive element), HTLV-I (human T-cell. leukemia virus I) Rexresponsive element or HTLV-II (human T-cell leukemia virus II) Rexresponsive element.
 23. A DNA sequence which codes for a reporterprotein and is operatively linked to a cis-active RNA export elementand, where appropriate, to a functional 5′ splice donor in the absenceof a functional 3′ splice acceptor, characterized in that (a) the DNAsequence coding for the reporter protein is modified at the nucleic acidlevel compared with the wild-type sequence, (b) RNA export for themodified DNA sequence takes place dependent on the cis-active RNA exportelement, and (c) essentially no RNA export for the wild-type DNAsequence takes place dependent on the cis-active RNA export element. 24.The DNA sequence as claimed in claim 23, characterized in that it isadditionally operatively linked to a transcription control sequence. 25.The DNA sequence as claimed in claim 24, characterized in that thetranscription control sequence comprises a constitutively activepromoter.
 26. The DNA sequence as claimed in claim 25, characterized inthat the constitutively active promoter is the CMV (cytomegalovirus) orthe SV40 (simian virus 40) promoter.
 27. The DNA sequence as claimed inclaim 23, characterized in that the transcription control sequencecomprises an inducible promoter.
 28. The DNA sequence as claimed inclaim 27, characterized in that the inducible promoter is atetracycline-dependent promoter.
 29. The DNA sequence as claimed in anyof claims 22 to 28, characterized in that it additionally comprises apolyadenylation signal.
 30. The DNA sequence as claimed in any of claims22 to 29, characterized in that the reporter protein is selected from afluorescent protein, a protein whose enzymatic activity is detectable,an immunologically detectable protein, a protein which serves asselection marker, or a transcription factor.
 31. The DNA sequence asclaimed in any of claims 22 to 30, characterized in that the cis-activeRNA export element is selected from MPMV CTE (Mason-Pfizer monkey virusconstitutive transport element), RSV CTE (Rous sarcoma virusconstitutive transport element), SRV CTE (simian retrovirus constitutivetransport element) or HIV-1 RRE (human immunodeficiency virus 1 Revresponsive element), HIV-2 RRE (human immunodeficiency virus 2 Revresponsive element), SIV RRE (simian immunodeficiency virus Revresponsive element), HTLV-I (human T-cell leukemia virus I) Rexresponsive element or HTLV-II (human T-cell leukemia virus II) Rexresponsive element.
 32. A eukaryotic cell, characterized in that it istransformed with a DNA construct as claimed in any of claims 22 to 31,where the DNA construct is present in a form capable of transcription.33. The eukaryotic cell as claimed in claim 32, characterized in that itexpresses one or more heterologous RNA export factors.
 34. Theeukaryotic cell as claimed in claim 33, characterized in that the RNAexport factors are viral proteins.
 35. The eukaryotic cell as claimed inclaim 34, characterized in that the RNA export factor is selected fromRev from HIV-1 (human immunodeficiency virus 1), HIV-2 (humanimmunodeficiency virus 2), SIV (simian immunodeficiency virus), EIAV(equine infectious anemia virus), Rex from HTLV-I (human T-cell leukemiavirus I) or HTLV-II (human T-cell leukemia virus II), or the 34K andE4orf6 proteins from adenoviruses, or EB2 proteins from EBV(Epstein-Barr virus), or ORF 57 gene products from herpesvirus saimiri,or ICP 27 proteins from HSC (herpes simplex virus).
 36. The eukaryoticcell as claimed in any of claims 32 to 35, characterized in that it istransformed with at least one nucleic acid which codes for a furtherreporter protein which can be detected in the presence of the reporterprotein for RNA export.
 37. A reagent kit comprising (a) a DNA sequenceas claimed in any of claims 23 to 31 and a cell which can be transfectedwith the DNA sequence or/and (b) a cell as claimed in any of claims 32to
 36. 38. The reagent kit as claimed in claim 37, additionallycomprising leptomycin B.
 39. A method for detecting a viral infection,characterized in that the activity of a viral nuclear export factor isdetermined.
 40. The method as claimed in claim 39, characterized in thatthe viral infection is an infection with a retrovirus.
 41. The method asclaimed in claim 39, characterized in that the viral infection is aninfection with a lentivirus.
 42. The method as claimed in claim 41,characterized in that the viral infection is an infection with HIV-1(human immunodeficiency virus 1) or HIV-2 (human immunodeficiency virus2).
 43. The method as claimed in claim 42, characterized in that theRev-dependent RNA export is detected.
 44. The method as claimed in anyof claims 39 to 43 for detecting viruses in a plasma sample.
 45. Themethod as claimed in claim 44, characterized in that a eukaryotic cellas claimed in claim 32 is used.
 46. A DNA sequence which codes for afluorescent protein, characterized in that the DNA sequence coding forthe fluorescent protein is modified at the nucleic acid level comparedwith the wild-type sequence in such a way that the selection of codonsis at least partially adapted to the preferred selection of codons of aretrovirus, in particular of a human retrovirus such as HIV-1.
 47. TheDNA sequence as claimed in claim 46, characterized in that it comprisesthe hivGFP sequence shown in FIG.
 1. 48. A DNA sequence comprising agene sequence coding for a fluorescent protein between a functional 5′splice donor and a functional 3′ splice acceptor.
 49. The use of the DNAsequence as claimed in claim 48 for investigating RNA export processes.50. The use of the DNA sequence as claimed in claim 48 for identifyingmodulators of RNA export from the cell nucleus of eukaryotic cells. 51.A method for detecting RNA export from the cell nucleus of eukaryoticcells, characterized in that a reporter protein which can be detectedwithout lysis or fixation of the cells is used.
 52. The method asclaimed in claim 51, characterized in that a fluorescent protein is usedas reporter protein.
 53. The method as claimed in claim 51,characterized in that a selection marker is used as reporter protein.54. The method as claimed in any of claims 51 to 53, characterized inthat (a) the cells are transfected or infected with a gene library (b)the genes which bring about a modulation of RNA export from the cellnucleus are purified by cultivating the cells and isolating the nucleicacid.